Unitary 3D culture device

ABSTRACT

A continuous device for culturing mammalian cells in a three-dimensional structure for the transplantation or implantation in vivo is described. The culturing device comprises (a) a scaffold formed by a matrix of interconnected growth surfaces spaced at regular intervals and (b) a fluid distribution means at the inlet and the exit of the growth areas. The device is particularly useful for culturing bone cells for dental implants or bone reconstruction.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.13/515,685 filed Oct. 23, 2012, now U.S. Pat. No. 9,220,731, which is a371 of PCT Patent Application No. PCT/EP2010/069768 filed Dec. 15, 2010,which in turn claims the benefit of European Patent Application No.09179465.1 filed Dec. 16, 2009, the contents of each of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a device for culturing mammalian cellsin a three-dimensional structure for the transplantation or implantationin vivo. More particularly, the present invention relates to acontinuous culturing device for culturing bone cells for dental implantsor bone reconstruction.

BACKGROUND OF THE INVENTION

There is increasing interest in growing cells in three-dimensional (3D)environments such as on a 3D structure or scaffold. Cell culture on 3Dscaffolds is useful in tissue engineering for the generation ofimplantable tissue structures. Intrinsic difficulties with 3D culturesin 3D scaffolds are (i) the uniform and efficient seeding of cellsthroughout the scaffold pores, and (ii) limited mass transfer to thecells in the central scaffold part.

The past three decades have shown great advances in the area of tissueengineering but the problem associated with the difficulty of culturingcells at the center of deep or thick structures remains.

U.S. Pat. No. 6,194,210 describes a process for hepatitis A virus in anaggregated microcarrier-cell culture.

U.S. Pat. No. 6,218,182 describes a method for culturing 3D tissues, inparticular liver tissue for use as an extracorporeal liver assistdevice, in a bioreactor where cells are seeded and provided with twomedia flows, each contacting a different side of the cells.

US 2009/0186412 describes a porous cell scaffold and methods for itsproduction.

All prior art references address the problems that arise when a culturesystem with a high density of cells encounters flow irregularities.

Known bioreactors do not efficiently simulate in vivo nutrient mechanismin thick structures or when culture density is high.

Regulation of flow, delivery of nutrients, gasses and removal of wastein the bodies of mammals is an automated process that encompasses manycomplex functions in the body. Blood is a complex system, that supportsthe ability to transport large quantities of gasses and nutrients to andfrom cells throughout the body. Flow is managed by a complex system thatautomatically alters volume and pressure to redistribute the flow ofblood to areas of high demand. The distribution system includesthousands of branches and each branch may have smaller internaldiameters until finally arriving at the dimensional level where thecells are nourished.

SUMMARY OF THE INVENTION

The use of Computational Fluid Dynamics (CFD) software permits analysisof the flow within a complex structure and its container. When asuitable combination of characteristics are identified, the metabolicparameters can be studied to assure that both the utilisation rate ofmaterials and the production of waste products remain in a typicallysafe zone. One example would be to calculate the maximum cell densityand the oxygen consumption rate, to assure that all the cells remainaerobic.

We have now found a continuous culture device which solves the problemof culturing cells at the center of deep or thick structures.

Object of the present invention is a continuous culture devicecomprising (a) a scaffold formed by a matrix of interconnected growthsurfaces spaced at regular intervals and (b) a fluid distribution meansat the inlet and the exit of the growth areas.

The spacing and definition are arranged to permit directional flowthrough and around the growth surfaces uniformly.

The fluid distribution means at the inlet and the exit of the growthareas permits an adequate flow to each growth surfaces. The fluiddistribution is analysed using computational fluid dynamics and keymetabolite utilisation analysis to assure that the cells are not subjectto detrimental growth conditions.

Preferably, the fluid distribution means distributes the incoming flowof fresh nutrients and gasses to the growth surfaces. Thecross-sectional area of the distribution device channels and the numberof channels can be adjusted to facilitate the uniform distribution tothe growth surfaces, depending on the shape of the growth surfaces andthe total number of cells supported by the growth surfaces.

Preferably, the culture device includes a matrix of interconnectedgrowth surfaces, defined by the interconnection of multiple fibers orthree-dimensional structures, in an organized and repetitive manner,which can incorporate any number of facets or surface artefacts utilisedto encourage or enhance the attachment and growth of cells.

The three-dimensional structures forming the matrix can be cylindrical,rectangular, hexagonal or any other shape or combination of shapes andthe surfaces may be smooth or textured.

In a practical preferred embodiment of the invention, the scaffold isformed by a matrix of interconnected growth surfaces spaced at regularintervals around a central support.

The open spaces formed by the interconnection of the structures, areequal or larger than 0.7 mm and smaller than 3 mm, preferably equal orlarger than 0.9 mm and smaller than 3 mm. The spacing in the preferredembodiment is greater than 1.0 mm, but can be altered as required by theneed for physical strength of the scaffold. In a still more preferredembodiment of the present invention, the interconnected growth surfacesare spaced at regular intervals equal or larger than 1.0 mm and lessthan 2.0 mm.

Spacing is a characterizing feature of the present invention. Thevariability of the parameter around the above range allows to optimizethe flow of medium throughout the scaffold and, at the same time, toimpart an adequate solidity to the 3D structure for all the devicesaccording to the invention independently from their final shape anddimension. The open spaces formed by the interconnecttion of the growthsurfaces create the organised characterizing structure of the device ofthe present invention which differs from the porous structure of thedevice known from the prior art.

The shape of the scaffold is preferably cubic but it could be anothershape, for example cylindrical or anatomically correct.

Preferably, the culture device includes a large number of interconnectedgrowth surfaces uniformly arranged to create large open areas that limitthe maximum number of cells per cubic volume facilitating the easyvascularization of the growth areas.

The culture device can be made of any biocompatible material.

Biocompatible materials are any biocompatible organic polymer or mixturethereof as well as blends or mixtures of biocompatible organic polymerswith biocompatible organic or inorganic non-polymeric compounds.

Non limitative specific examples of components of the biocompatiblematerial useful in the present invention are polycaprolacton,polyethylene oxide-terephthalate, polyamide, poly-L-lactic acid,polyglycolic acid, collagen, fibronectin, hydroxyapatite, etc.

In a practical preferred embodiment, the culture device furthercomprises an aseptically sealed housing that can be disassembled at thecompletion of the culture period. Said aseptic housing can include asealed removable cover, an inlet distribution means, an optional exitdistribution means, and the necessary support means required to locateand secure the growth surfaces in the culture device.

The housing can be in the form of a rectangle, cylinder or any othershape necessary to hold the culture device and provide additionalfeatures for aseptic removal of the scaffold. The present inventionoffers several advantages over previous culture devices in that nutrientdelivery permits the creation of and maintaining the viability of tissueon a thick (>1 mm) substrate.

The 3D culture device of the present invention can be produced in asingle step process. Alternatively, a 2D layer can be produced first,and then the single 2D layers can be assembled one over the others toform the 3D culture device according to the present invention.

The final dimension of the 3D culture device will depend on the numberof assembled 2D layers.

The culture device of the present invention can be efficiently used forculturing any kind of cells into a 3D tissue. Preferably it is used forculturing cells for dental implants or bone reconstruction. Once thecells have grown into a 3D tissue, the media flow may be stopped and thetissue can be used or preserved for future use.

The culture device of the present invention can be efficiently used alsofor culturing cells directly into the body. In fact, the device can beimplanted into the patient in need of tissue reconstruction and theculturing is effected in vivo.

By using the culture device according to the present invention cells maybe grown in a controlled environment on a biodegradable scaffold. Thelarge open areas formed by the interconnection of the growth surfacesallows them to be exposed to a uniform flow of medium and to preventfouling during the growth process. In particular, fouling or blockade ofthe growth surface by gas bubbles during the growth process isprevented. Moreover, with the culture device of the invention, cultureconditions are monitored continuously and any departures from thedesired conditions are automatically corrected and alarmed. Thisprovides conditions necessary to maintain cells in theirundifferentiated state, to minimise the maximum cell density and theassociated toxic necrosis, and to provide an environment that is notdiffusion limited for key nutrients and gasses.

Furthermore the culture device according to the present inventionprovides the growth of tissues also in the absence of cells, as shown inexperiments carried out on rabbits.

EXAMPLE Experimental Protocol

A two-layer scaffold (11 mm×11 mm×5 mm) according to the presentinvention, cut in four pieces of equal dimension, was used for the cellgrowth experiment on mice.

Histological Analysis and Results

The four continuous culturing devices were implanted intoimmunodeficient NOD/SCID mice.

The analysis of the inflammatory reaction after one week from theimplantation showed no sign of typical inflammatory reaction, i.e.swelling, redness, exudates, etc.

Histological analysis of the control material HA (Hydroxy Apatite), i.e.a biomedical material commercially available used as standard sample,did not reveal phlogosis (e.g. lymphocytic infiltration) conversely itrevealed the integration of the porous ceramic material with the tissues(fibroblast colonization of the material's pores).

Poly-capro-lactone was removed from all the samples containing thecontinuous culturing device object of the present invention and wasreplaced by paraffin. It resulted in a negative or empty image onmicrophotographs.

Histological analysis of the samples P (Polycaprolactone), PC(Polycaprolactone with cells), PD (Polycaprolactone with tri-calciumphosphate dipping) (FIG. 7), and PDC (Polycaprolactone with tri-calciumphosphate dipping with cells) (FIG. 8) did not reveal any tissue'sinflammatory process. Then the implantation in vivo of the continuousculturing device object of the present invention, provided cell growthwithout involving tissue's inflammation process.

The analysis carried out on mice demonstrated that the continuousculturing device is biocompatible and not locally toxic. Moreover thecharacteristic 3D structure of the continuous culturing device providesthe tissue's regrowth.

The present invention is now illustrated in more details in thefollowing drawings which represent specific embodiments of the inventionwithout limiting it.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 One embodiment of the scaffold

FIG. 2 One embodiment of a flow distribution device

FIG. 3 One embodiment of scaffold between two flow distribution devices

FIG. 4 System flow chart

FIG. 5 CFD Flow analysis

FIG. 6 Photographic Flow analysis

FIG. 7 Microphotograph of the sample PD

FIG. 8 Microphotograph of the sample PDC

FIG. 9 Photographs of samples HA, PD and PDC

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is one embodiment of the scaffold. Scaffold (1) is formed by theinterconnection of a matrix of cylindrical (3) structures. The scaffold(1) is formed around the central support (2).

FIG. 2 is one embodiment of the fluid distribution device (5). In thisdevice, the fluid is presented to the device (5) at a common conduit (6)which is connected to the distribution conduits (7). A support means (8)is shown to connect with the central support (2) of the scaffold (1).

FIG. 3 depicts a scaffold (1) positioned between two of the distributiondevices (5). In this embodiment the fluid is delivered to the inletcommon conduit (6) and further distribute to distribution conduits (7)and then is distributed through and around the open structures (4) ofscaffold (1). The fluid is then collected and presented to commonconduits (7), located in the outlet device (5B) where it is collectedand presented to the common conduit (6) of the distribution device (5B).

FIG. 4 is an outline view of the culture device (10) connected to acentral circulation system (9). When the culture device (10) isconnected to system (9), it is positioned to receive a continuous flowof nutrients and dissolved gasses provided by pump (12). A centralcirculation loop is created by connecting the outlet of pump (12) withthe inlet of the culture device (10). The outlet of the culture device(10) is connected with the inlet of pump (12) through the fluidreservoir (13). In constant communication with the fluid in the system(12) are a variety of sensors (11). The sensors (11) are connected witha control means (20) that monitors and controls the conditions of system(12). Additional pumps (14, 15) are provided to supply metered deliveryof fresh nutrients to the system, and waste materials from the system.

FIG. 5 illustrates an example of Computational Fluid Dynamics analysis,where the distribution of flow is throughout the structure.

FIG. 6 is a photographic flow analysis.

FIG. 7 and FIG. 8 illustrate the growth of cells and the absence ofinflammatory process for the sample PD and the sample PDC respectively.Microphotographs are 20× of magnification and the cutis are on the topof the microphotographs.

FIG. 9 illustrates the areas of application and analysis of the samplesHA, PD and PDC for the cells growth experiments on mice. The externalanalysis of the samples does not reveal any fibrotic reaction orinflammation process.

The invention claimed is:
 1. A unitary 3D culture device comprising: ascaffold comprised of 3D lattice-shaped growth structures forming amatrix of interconnected orthogonal growth surfaces spaced along andparallel to x, y, and z Cartesian axes, the growth surfaces forming amatrix of cubic open spaces arranged parallel to the x, y, and zCartesian axes; an inlet fluid distribution device connected to an inputside of the scaffold and having a common fluid intel that is fluidlycoupled with a plurality of inlet distribution conduits through whichinput fluid is delivered to and distributed through the open spaces ofthe scaffold; an outlet fluid collection device connected to an outputside of the scaffold and having a plurality of outlet conduits that arefluidly coupled with a common fluid outlet that collects output fluidfrom the open spaces of the scaffold; a housing coupled to the inletfluid distribution device and the outlet fluid collection device, andthat houses the scaffold, inlet fluid distribution device, and outletfluid collection device; and wherein the housing, scaffold, inlet fluiddistribution device, and outlet fluid collection device comprise asingle work piece.
 2. The device of claim 1, wherein the open spaceshaving dimensions from 0.7 mm to 3 mm.
 3. The device of claim 2, whereinthe open spaces have dimensions from 1 to 2 mm.
 4. The device of claim1, wherein the scaffold is made of a biocompatible material.
 5. Thedevice of claim 4, wherein the biocompatible material includes at leastone material selected from the group consisting of polycaprolacton,polyethylene oxide-terephthalate, polyamide, poly-L-lactic acid,polyglycolic acid, collagen, fibronectin, and hydroxyapatite.
 6. Thedevice of claim 1, wherein the housing comprises an aseptically sealedhousing.
 7. The device of claim 1, wherein the housing comprises aremovable cover.
 8. The device of claim 1, wherein the housing comprisesat least one of the following forms: a rectangular form, and acylindrical form.
 9. The device of claim 1, wherein the inlet fluiddistribution device is configured to couple with an inlet pump via thecommon fluid inlet through the housing.
 10. The device of claim 1,wherein the outlet fluid collection device is configured to couple withan outlet pump via the common fluid collection via the common fluidoutlet through the housing.
 11. The device of claim 1, furthercomprising multiple 2D layers assembled one over another forming theunitary 3D culture device.
 12. The device of claim 1, wherein thescaffold comprises a cubic shape.
 13. The device of claim 1, wherein thescaffold comprises a cylindrical shape.
 14. The device of claim 1,wherein the scaffold comprises an anatomically correct shape.
 15. Thedevice of claim 1, wherein the growth structures comprise solidcylindrical structures.
 16. The device of claim 1, wherein the growthstructures comprise fibers.
 17. The device of claim 1, wherein thescaffold further comprises a central support.
 18. The device of claim17, wherein the inlet fluid distribution device couples to the scaffoldvia the central support at the input side of the scaffold.
 19. Thedevice of claim 17, wherein the outlet fluid collection device couplesto the scaffold via the central support at the output side of thescaffold.